Phosphorylation of Arabidopsis response regulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7 to act as a negative regulator of cytokinin signaling

Cytokinins are plant hormones that regulate diverse aspects of plant growth and development. Arabidopsis cytokinin signal transduction utilizes a multi-step two-component signaling (TCS) system by histidyl–aspartidyl phosphorelays. We here show that phosphorylation of ARR7, an A-type response regulator that acts as a negative regulator of cytokinin signaling, is required for its function in plants. Phosphorylation of ARR7 is inhibited in vitro by mutation in a putative phospho-accepting Asp residue into an Asn residue (ARR7^D85N). While ectopic expression of ARR7 decreases root-growth inhibition, callus formation, and cytokinin-inducible gene expression, overexpression of ARR7 ^D85N at the similar level does not generate these phenotypes. ARR7^D85N is localized to the nucleus and the half-life of this mutant protein is similar to that of ARR7 in Arabidopsis mesophyll protoplasts. These results suggest that the phosphorylation of ARR7 is necessary for ARR7-mediated cytokinin response.